Molecular Mechanisms Regulating Pancreatic Delta Cell Function and Dysfunction

项目摘要 2型糖尿病患者和动物胰岛葡萄糖刺激的生长抑素(SST)分泌缺失 这种疾病的模型会导致胰升糖素和胰岛素分泌中断。这是被普遍接受的 细胞的SST分泌是对细胞内钙离子升高的反应，这主要是由于 内质网(内质网)钙(Ca~(2+))释放。然而，控制-细胞内钙内质网处理的机制 它们在T2D中是如何改变的，在很大程度上是未知的。我们实验室的数据发现，富含胰岛的两孔- 结构域K+通道Talk-1是一种内质网局部通道，它为细胞内钙内质网的释放提供逆流 和钙离子内质网泄漏。Talk-1介导的细胞Ca~(2+)内流泄漏电化学驱动力的增强 菌株的钙内质网储存，限制葡萄糖刺激的钙内质网释放和SST分泌。进一步的数据显示， 葡萄糖诱导的-细胞钙-感受器的变构激活可使-细胞内钙内质网的释放和SST的分泌增加。 ING受体(CASR)。最后，我们的初步数据提供了糖尿病病情减轻的第一个证据 -细胞内钙内质网储存，参与葡萄糖刺激的钙处理和海绵体分泌的扰动 在糖尿病的情况下。基于这些令人振奋的初步数据，这项提案的总体目标是 阐明在糖尿病的发病机制中，-细胞钙内质网是如何被控制和破坏的。这个项目 将检验中心假设，即葡萄糖刺激的细胞的Sst分泌是由CaSR介导的钙内质网放大的 释放，受钙内质网存储的Talk-1通道约束控制。这背后的理由是 项目是了解CaSR和Talk-1如何控制-细胞内钙内质网的处理和SST的分泌将暴露 T2D中恢复葡萄糖刺激的SST分泌和胰岛激素分泌的新治疗靶点。这 该项目将以以下两个具体目标完成：1)确定-cell CaSR如何控制钙内质网 处理、SST分泌和胰岛激素分泌；以及2)决定Talk-1如何控制钙内质网 释放调节细胞的功能和功能障碍。在第一个目标下，将细胞去除的转基因小鼠 CASR以及具有细胞CaSR的shRNA敲除的人类伪胰岛将被用来评估其作用 钙感受受体在分泌调节细胞的钙离子处理和海绵体分泌过程中的作用。Aim1将 同时确定糖尿病条件下细胞钙内质网储备库的耗尽如何影响钙激活受体信号和体温。 分泌物。在第二个目标下，功能-1\f25 Talk-1\f25 Channels on-1\f25 Ca+ER-1\f6的处理和功能将是 在细胞特异性消融Talk-1的小鼠和含有细胞的人假性胰岛中检测到 下调Talk-1或表达显性负性Talk-1通道亚单位。此外，AIM2将 确定在应激条件下Talk-1如何增加细胞钙内质网耗竭 糖尿病导致细胞功能障碍。这个项目意义重大，因为它有望照亮机械- 在T2D中改变-细胞钙内质网处理和扰乱胰岛激素分泌的NIMS。此外，这个项目将 确定使T2D患者SST分泌正常化和减少胰岛功能障碍的药物策略。

Project Summary Islet glucose-stimulated somatostatin (Sst) secretion is lost in patients with type-2 diabetes (T2D) and in animal models of the disease, which contributes to disrupted glucagon and insulin secretion. It is generally accepted that Sst secretion from -cells occurs in response to elevated intracellular Ca2+, which primarily results from endoplasmic reticulum (ER) Ca2+ (Ca2+ER) release. However, the mechanisms that control -cell Ca2+ER handling and how they are altered in T2D are largely unknown. Data from our lab finds that the islet-enriched two-pore- domain K+ channel, TALK-1, is an ER localized channel in that provides a countercurrent for -cell Ca2+ER release and Ca2+ER leak. TALK-1-mediated augmentation of the electrochemical driving force for -cell Ca2+ER leak con- strains Ca2+ER storage, which limits glucose-stimulated Ca2+ER release and Sst secretion. Further data show that -cell Ca2+ER release and Sst secretion are amplified by glucose-induced allosteric activation of -cell Ca2+-sens- ing receptors (CaSRs). Finally, our preliminary data provide the first evidence that diabetic conditions diminish -cell Ca2+ER storage, which contributes to perturbations in glucose-stimulated Ca2+ handling and Sst secretion under diabetic conditions. Based on these exciting preliminary data, the overall objective of this proposal is to elucidate how -cell Ca2+ER is controlled and becomes disrupted during the pathogenesis of diabetes. This project will test the central hypothesis that glucose-stimulated -cell Sst secretion is amplified by CaSR-mediated Ca2+ER release, which is controlled by TALK-1 channel constraint of Ca2+ER storage. The rationale that underlies this project is that understanding how CaSR and TALK-1 control -cell Ca2+ER handling and Sst secretion will expose novel therapeutic targets for restoring glucose-stimulated Sst secretion and islet hormone secretion in T2D. This project will be accomplished with the following two specific aims: 1) Determine how -cell CaSR controls Ca2+ER handling, Sst secretion, and islet hormone secretion; and 2) Determine how TALK-1 channel control of Ca2+ER release modulates -cell function and dysfunction. Under the first aim, transgenic mice with -cell ablation of CaSR as well as human pseudoislets with ShRNA knockdown of -cell CaSR will be utilized to assess the roles of the Ca2+-sensing receptor during secretagogue modulation of -cell Ca2+ handling and Sst secretion. Aim1 will also determine how depletion of -cell Ca2+ER stores under diabetic conditions impacts CaSR signaling and Sst secretion. Under the second aim, the function TALK-1 channels on -cell Ca2+ER handling and function will be determined in mice with -cell specific ablation of TALK-1 and in human pseudoislets containing either -cells with knockdown of TALK-1 or expressing dominant negative TALK-1 channel subunits. Furthermore, Aim2 will determine how TALK-1 augmentation of -cell Ca2+ER depletion under the stressful conditions associated with diabetes contributes to -cell dysfunction. This project is significant because it is expected to illuminate mecha- nisms that alter -cell Ca2+ER handling and disrupt islet hormone secretion in T2D. Moreover, this project will identify pharmacological strategies for normalizing Sst secretion and reducing islet dysfunction in T2D.